Oxidative stress adaptation with acute, chronic, and repeated stress

Free Radical Biology and Medicine 55 (2013) 109–118

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Original Contribution

Oxidative stress adaptation with acute, chronic, and repeated stress Andrew M. Pickering a,b, Lesya Vojtovich a, John Tower b, Kelvin J. A. Davies a,b,n a

Ethel Percy Andrus Gerontology Center, Davis School of Gerontology, and Molecular and Computational Biology Program, Department of Biological Sciences, Dornsife College of Letters, Arts & Sciences, University of Southern California, Los Angeles, CA 90089, USA b

a r t i c l e i n f o

abstract

Article history: Received 29 May 2012 Received in revised form 7 October 2012 Accepted 2 November 2012 Available online 9 November 2012

Oxidative stress adaptation, or hormesis, is an important mechanism by which cells and organisms respond to, and cope with, environmental and physiological shifts in the level of oxidative stress. Most studies of oxidative stress adaption have been limited to adaptation induced by acute stress. In contrast, many if not most environmental and physiological stresses are either repeated or chronic. In this study we find that both cultured mammalian cells and the fruit fly Drosophila melanogaster are capable of adapting to chronic or repeated stress by upregulating protective systems, such as their proteasomal proteolytic capacity to remove oxidized proteins. Repeated stress adaptation resulted in significant extension of adaptive responses. Repeated stresses must occur at sufficiently long intervals, however (12-h or more for MEF cells and 7 days or more for flies), for adaptation to be successful, and the levels of both repeated and chronic stress must be lower than is optimal for adaptation to acute stress. Regrettably, regimens of adaptation to both repeated and chronic stress that were successful for short-term survival in Drosophila nevertheless also caused significant reductions in life span for the flies. Thus, although both repeated and chronic stress can be tolerated, they may result in a shorter life. & 2012 Elsevier Inc. All rights reserved.

Keywords: Oxidative stress Ubiquitin–proteasome system Protein degradation Repeated stress Chronic stress Stress adaptation Free radicals

Introduction Aerobic organisms must live with a multitude of free radicals and related reactive oxygen/nitrogen species. Perhaps the most ubiquitous of these species is hydrogen peroxide (H2O2), which is found at measurable levels in all animal tissues. At nanomolar levels, H2O2 is a stimulant of cell growth and proliferation, whereas micromolar levels cause transient growth arrest and induce protective adaptive alterations in gene expression [1–4]. At millimolar levels, and above, H2O2 is clearly a toxic oxidant, causing frank oxidative stress. One of the major results of such oxidative stress is modification of cellular proteins, which can block enzymatic activities, inhibit normal cell functions, and even cause cellular apoptosis or necrosis [5–13]. As a primary defense against oxidative stress, cells possess a range of antioxidant compounds and enzymes that function to keep the levels of free radicals and reactive oxidants low [14–17]. Although these

Abbreviations: MEF, murine embryonic fibroblast; AMC, 7-amino-4-methylcoumarin; Suc-LLVY-AMC, the succinylated peptide N-succinyl-leucine-leucinevaline-tyrosine-7-amino-4-methylcoumarin (used as a peptide substrate to estimate proteolytic capacity); PBS, phosphate-buffered saline; Hb, hemoglobin; Hbox, oxidatively modified hemoglobin n Corresponding author at: Ethel Percy Andrus Gerontology Center, Davis School of Gerontology, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089, USA Fax.: þ (213) 740-6462. E-mail address: [email protected] (K.J. A. Davies). 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.11.001

primary antioxidant defenses are sufficient under conditions of low or normal oxidant levels, they are overwhelmed during periods of oxidative stress. This results in oxidative protein damage, which, if not removed quickly, can accumulate over time and cause deterioration of cell function. It is the role of proteases, such as the 20S proteasome, the immunoproteasome [3,4,18–28], and the mitochondrial Lon protease [29–32] (as part of a secondary antioxidant response system), to rapidly and selectively remove such damaged proteins, thus preventing their aggregation, cross-linking, and accumulation, as well as the cellular toxicity that would otherwise result. The degree of oxidative stress to which organisms are exposed is not static but shifts with changes in environment, pollution, metabolism, diet, lifestyle, and age. Both eukaryotic cells and prokaryotes have highly dynamic responses to these changes in oxidative stress levels. It seems that all life forms can transiently adapt to temporary changes in stress levels, by a combination of direct enzyme activation/deactivation and alterations in the level of expression of more than a hundred genes [2–4,33]. Such transient adaptation is also called hormesis [34,35]. Mammalian cells, including humans, can shift their patterns of gene expression over an 18-h period such that housekeeping genes, growth, and proliferation are suppressed, and protective genes are overexpressed; the adapted cell is then significantly more resistant to oxidative stress. If the stress is removed or declines during this 18-h period, cells will stay adapted for only another 6–12 h (approximately 24–30 h in all), after which they gradually de-adapt. Although there are many

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important enzymes that contribute to this adaptive response, we have previously demonstrated that increased synthesis of the 20S proteasome, the immunoproteasome, and the Lon protease is required for efficient clearance of oxidatively damaged proteins and for greatest stress protection [2–4,33]. Most of the work to date on transient adaptation to oxidative stress has involved experiments limited to a single acute stress exposure. In previous work, using a wide range of oxidants and cell types, we have described a pretreatment and challenge model to assay oxidative stress adaptation [2–4]. In this system cells were exposed to a single, mild, nontoxic dose of an oxidant such as H2O2 and then permitted to adapt for a period of time (typically 18–24 h). The cells were then exposed to a much higher dose of the oxidant, termed the challenge dose, which would normally be toxic without the pretreatment and adaptive period. We found that pretreated cells are considerably more resilient to the toxic challenge level of oxidative stress than are nonpretreated cells. In contrast, as discussed above, both metabolic and environmental sources of oxidative stress are highly variable and can also occur repeatedly or even continuously for prolonged periods. Thus, it is important that we develop an understanding of stress-adaptive capacities under both repeated and chronic stress conditions that may better reflect real-life experiences. In this study we have examined the ability of both mammalian cells and the invertebrate model organism Drosophila melanogaster to adapt to oxidative stress under an acute oxidative stress exposure and compared it to a more environmentally representative stress of repeated and chronic oxidative stress exposure. In addition we examined the long-term repercussions of such oxidative stress adaptation.

Materials and methods Materials All materials were purchased from VWR unless otherwise stated. Murine embryonic fibroblasts (MEFs)1, Cat. No. CRL-2214, were purchased from the ATCC (Manassas, VA, USA). Cells were grown in Dulbecco’s modified Eagle’s medium, Cat. No. 10-013-CV, from Mediatech (Manassas, VA, USA) and supplemented with 10% fetal bovine serum (Cat. No. SH30070.03) from Hyclone (Logan, UT, USA): henceforth referred to as ‘‘complete medium.’’ Cells were typically incubated at 371C under 5% CO2 and ambient oxygen. Adaptation to oxidants MEF cells were grown to 10% confluence (E100,000 cells/ml) and then pretreated with H2O2. The optimum pretreatment condition, causing maximum adaptation, was found to be 1 nmol of H2O2 per 105 cells (1 mM final concentration; Cat. No. H1009-100ml; Sigma–Aldrich, St. Louis, MO, USA), for 1-h at 371C under 5% CO2. Cells were then washed once with phosphate-buffered saline (PBS), which was finally replaced with fresh complete medium. Fluoropeptide proteolytic assays Preparation of mammalian cells MEFs were harvested by scraping in phosphate buffer. Cells were then resuspended in 50 mM Tris, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT) (pH 7.5) and lysed by three freeze–thaw cycles. Preparation of Drosophila Ten flies were collected per sample. The flies were then transferred into a solution containing proteolysis buffer (50 mM

Tris, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM DTT, at pH 7.5). Flies were frozen once, then homogenized using a pestle, after which lysis was performed by three freeze–thaw cycles on dry ice for 5 min each, followed by a room temperature water bath for 5 min. The samples were then centrifuged at 10,000g to remove cuticle fragments and unlysed cells. Proteolytic assays Protein content was quantified using a BCA protein assay kit. Samples were transferred, in triplicate, in 5.0-mg cell lysate aliquots to 96-well plates, and 2 mM N-succinyl-Leu-Leu-Val-Tyr-AMC (Cat. No. 80053-860), purchased from VWR (Chester, PA, USA), was added to all samples in the plate. Plates were incubated at 37 1C and mixed at 300 rpm for 4-h. Fluorescence readings were taken at 10-min intervals using an excitation wavelength of 355 nm and an emission of 444 nm. Fluorescence units were converted to moles of free AMC, with reference to an AMC standard curve of known amounts of AMC (Cat. No. 164545) purchased from Merck (Whitehouse Station, NJ, USA). Background readings were then subtracted. Preparation of AMC-labeled hemoglobin and AMC-labeled oxidized hemoglobin Hemoglobin was labeled with the fluorophore AMC (to produce Hb–AMC) as described by Pickering and Davies [40], using 5 mg/ml hemoglobin labeled with 1 mM AMC (Cat. No. 164545) purchased from Merck. Next, some samples were incubated with 1 mM H2O2 for 1-h to create AMC-labeled oxidized hemoglobin (Hbox–AMC). Samples were incubated for a further 1-h and then extensively dialyzed to remove unbound AMC. Samples were also buffer-exchanged against proteolysis buffer (50 mM Tris, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM DTT, pH 7.5). After buffer exchange, BCA assays were used to adjust the concentration of the protein substrates. Hb–AMC or Hbox–AMC proteolytic assays MEF cells were harvested by cell scraping in phosphate buffer. The cells were then resuspended in 50 mM Tris, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM DTT (pH 7.5) and lysed by three freeze– thaw cycles. Protein content was quantified using a BCA protein assay kit. Then 5.0 mg of cell lysate per sample was transferred in triplicate to 96-well plates, and 5 ml of either Hb–AMC or Hbox– AMC was added to each plate. Plates were incubated at 371C and mixed at 300 rpm for 4-h. Fluorescence readings were taken at 10-min intervals using an excitation wavelength of 355 nm and an emission of 444 nm. Fluorescence units were converted to moles of free AMC, with reference to an AMC standard curve of known amounts of AMC (Cat. No. 164545) purchased from Merck. Background readings were then subtracted. siRNA (short interfering RNA) knockdown treatments

b1 (sc-62865) and scrambled (sc-29528) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For experiments with these siRNAs, MEF cells were seeded at a density of 105 cells/well in six-well plates and grown to 10% confluence. siRNA treatment was then performed over a 48-h period as described in the Santa Cruz Biotechnology product manual. Cell counting assay Cells were seeded in 100 ml samples and grown to 20% confluence in 48-well plates. Then, 24-h after seeding, some cells were pretreated with hydrogen peroxide or glucose oxidase (Cat. No. 700011-406) purchased from VWR for 1- or 24-h, depending on the

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assay. Samples were then washed with PBS, and new medium was added. At 24-h after treatment, cells were challenged with a toxic dose of 500 mmol of H2O2 per 105 cells (1 mM H2O2 final concentration) for 1-h, followed by addition of fresh complete medium. Cells were harvested 24-h after challenge, using trypsinization. The cell density of 100-ml samples of cell suspensions was then obtained in triplicate using a cell counter purchased from Beckman Coulter (Fullerton, CA, USA). BrdU (bromodeoxyuridine) assay for DNA replication and cell division BrdU, a synthetic thymidine analogue, can be incorporated into newly synthesized DNA providing a test of DNA replication, as an indirect measure of cell division. The assay was performed as described in the product manual from Millipore. BrdU incorporation was detected by addition of peroxidase substrate. Spectrophotometric detection was performed at a wavelength of 450 nm. OxyBlot assay for protein carbonyls (protein oxidation) An OxyBlot kit for detection of protein carbonyls was purchased from Millipore and assays were performed as described in the product manual. D. melanogaster culture Drosophila were cultured on a standard agar/dextrose/corn meal/yeast medium [48] at 25 1C. Unless otherwise stated, w[1118] flies were used in all assays. Flies were collected over a 48-h period from precleared bottles and allowed 4 days to mature so that at initiation of assays, flies were 4–6 days of age. All assays were performed exclusively on female flies. Drosophila H2O2 adaptation Samples of 20 flies were transferred to vials containing ½ a Kim-Wipe soaked in 1 ml of 5% sucrose plus 0 mM, 10 mM, 100 mM, or 1 mM H2O2 for 8-h. Flies were then returned to normal vials for 16-h to permit adaptation to occur. The flies were then challenged with a toxic dose of H2O2. This procedure has previously been shown to be an effective method of exposing flies to oxidative stress [43]. Drosophila RNAi experiments Flies expressing RNAi against a required proteasome subunit, prosb2RNAi (w[1118]; P(GD10938)v24749) were purchased from the Vienna Drosophila RNAi Center (Vienna, Austria). Males from this line (or w[1118] as a control) were crossed with virgin females containing the Act-GS-255B driver [45,49]. Parents were removed 4 days after initiation of cross. Progeny were then collected over a 48-h period after eclusion. The Act-GS-255B driver was activated by feeding flies RU486. Flies were cultured in normal vials containing either 50 ml of stock RU486 (20 mg/ml) or ethanol, which had been added to vials and air-dried 24-h before the assay. Flies were incubated in these vials for 2 days. For H2O2 adaptation, the flies were then pretreated by exposure to ½ a Kim-Wipe soaked in 1 ml of 5% sucrose 7 100 mM H2O2 for 8-h and then returned to RU486 or ethanol vials for a further 16-h of RU486 treatment. Drosophila H2O2 challenge assays Samples of 20 flies were transferred to vials containing ½ a Kim-Wipe soaked in 1 ml of 5% sucrose and 4.4 M H2O2. Survival

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was then scored ever 8-h after initiation of challenge. Flies were scored as dead once they became completely immobile.

Results Transient adaptive response to acute H2O2 pretreatment in MEF cells For baseline data on adaptation to a single H2O2 pretreatment, MEF cells were incubated with 1 nmol of H2O2 per 105 cells (1 mM H2O2 final concentration), which was found to be the optimum pretreatment condition in preliminary experiments. Cells were then allowed to adapt for 24-h and then challenged for 1-h with 500 mmol of H2O2 per 105 cells (1 mM H2O2 final concentration), which was optimized to be sufficiently strong to cause almost complete growth arrest, yet with minimal apoptosis. Adaptation was then scored using a variety of techniques, including cell counts 24-h after challenge (Fig. 1A) and BrdU incorporation over the 24-h period after H2O2 challenge (Fig. 1B). In both of these experiments, H2O2 challenge caused a strong decrease in cell division, which was partially prevented by H2O2 pretreatment. We next tested whether the pretreatment-induced resistance to H2O2 challenge involved less of an accumulation of oxidatively damaged proteins, as measured by a protein carbonyl assay. H2O2 challenge induced almost a doubling of protein carbonyl content, but this was cut in half if the cells were first pretreated with H2O2 24-h before challenge (Fig. 1C). In previous reports, the proteolytic capacity of cellular 20S proteasome has been suggested to be important for surviving an oxidative stress [3,4,18,33,36,37]. It has been shown for instance that much of the adaptive increase in proteolytic capacity and oxidative stress tolerance is lost by blocking new synthesis of the 20S proteasome [3,4,38,39]. We, therefore, tested whether the observed increase in oxidative stress tolerance was dependent on 20S proteasome. To do this we briefly exposed cells to siRNA against the b1 subunit of the 20S proteasome 48-h before H2O2 pretreatment. We found that this treatment completely abolished the H2O2 pretreatmentinduced increase in cell counts, and BrdU incorporation, as well as the H2O2 pretreatment-induced decrease in protein carbonyl content (data not shown). We reasoned that the adaptive response to oxidative stress should be transient and that oxidative stress tolerance should return back down to normal levels once the stimulatory effect of the H2O2 pretreatment had abated. To test this we ran a cell count time course for oxidative stress tolerance for up to 48-h after H2O2 pretreatment. Our results show that the adaptive response was indeed transient and reversible over a 48-h period (Fig. 2A). At 24-h after pretreatment, cells had a heightened oxidative stress tolerance, but 48-h after pretreatment their resistance had returned to baseline levels. Because of the important role of proteolysis in oxidative stress adaptation we also measured proteolytic capacity. To test this, in the current model, we measured proteolytic capacity against the fluoropeptide SucLLVY-AMC, which is commonly used as a marker for the chymotrypsin-like activity of the proteasome. We found that proteolytic capacity gradually increased in the first 24-h after H2O2 pretreatment, to more than double baseline levels, but then (like overall H2O2 tolerance), it declined over the following 48-h (Fig. 2B). Adaptive responses to multiple H2O2 pretreatments in MEF cells From the results of Figs. 1 and 2, we conclude that a mild, acute H2O2 pretreatment transiently improves oxidative stress tolerance. As pointed out in the introduction, however, environmental and metabolic oxidative stress exposures are often repeated or even

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Fig. 1. H2O2 pretreatment increases tolerance to H2O2 challenge. H2O2 pretreatment enhances resistance to subsequent H2O2 challenge using three different measures of oxidative stress tolerance (cell counts, BrdU incorporation, and protein carbonyl content). In all cases, MEF wells were grown to 10% confluence, then some samples were pretreated with a mild adaptive dose of H2O2, which was 1 nmol of H2O2 per 105 cells (1 mM H2O2 final concentration), for 1-h. Cells were then challenged 24-h later with a toxic dose of H2O2, which was 500 mmol of H2O2 per 105 cells (1 mM H2O2 final concentration), for 1-h. (A) After challenge, cells were permitted to divide for 24-h, then cell counts were taken. (B) After H2O2 challenge BrdU was added to cell cultures, cells were permitted to divide for 24-h, then BrdU incorporation was measured. (C) Protein carbonyl content was measured 6-h after H2O2 challenge. (D, E) Cells were treated with either scrambled or b1 siRNA for 48-h and then pretreated and assayed as for (A–C). In all cases values represent means 7 SE, where n ¼ 3.

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Fig. 2. H2O2 pretreatment causes a transient adaptive response. H2O2 pretreatment transiently enhances resistant to subsequent H2O2 challenge and increases cellular proteolytic capacity, but cells return to a naı¨ve state 48–72-h after H2O2 pretreatment. (A) With H2O2 pretreatment oxidative stress tolerance increases for 24-h and then returns to baseline levels. Cells were prepared and pretreated as for Fig. 1. The cells were then challenged either 24- or 48-h after pretreatment. Cell counts were taken 24-h after challenge. Values are plotted as a percentage of the cell counts of samples that were not exposed to the challenge and are shown as means 7 SE, where n ¼ 4. The values for unchallenged samples and 0-h pretreated samples represent data pooled from cell counts taken 24- and 48-h after pretreatment. (B) With H2O2 pretreatment, proteolytic capacity increases in the 24-h after pretreatment but returns to baseline levels over the succeeding 72-h. Cells were prepared and pretreated as for Fig. 1. Samples were then collected 12-, 24-, 48-, and 72-h after H2O2 pretreatment. Samples were then lysed and proteolytic capacity was determined through degradation of Suc-LLVY-AMC. Values are means 7 SE, where n ¼ 3. The values for 0 h pretreated samples represent the activity of non-pretreated cells taken 12-h after H2O2 pretreatment.

chronic in nature. To begin experiments of changes in proteolytic capacity under repeated stress, we first exposed cells to the adaptive dose of 1 nmol of H2O2 per 105 cells and measured proteolytic capacity 24-h later, through degradation of the chymotrypsin-like substrate Suc-LLVY-AMC (Fig. 3A). Repeated oxidative stress was then initiated by exposing cells to the adaptive dose of H2O2 a second, or even a third, time after the initial exposure, at intervals of 3-, 6-, or 12-h after the initial exposure. We observed that a single

pretreatment caused an increase in proteolytic capacity. If cells were pretreated for a second time 12-h after the first treatment, the second pretreatment was tolerated and proteolytic capacity actually increased slightly more than with a single pretreatment. If, however, samples were pretreated a second or even a third time within 6-h of the initial pretreatment, then the proteolytic capacity was lower than that seen with a single pretreatment, but still greater than that of naı¨ve (non-pretreated) cells. When cells were given second and

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Fig. 3. Capacity to degrade Suc-LLVY-AMC following repeated H2O2 pretreatments. (A) Additional H2O2 treatments 3- or 6-h after the initial treatment blunt the adaptive increase in proteolytic activity, whereas repeated treatments 12-h after the initial treatment seem to be tolerated. Cells were prepared and pretreated as for Fig. 1. Some cells were then pretreated with the same dose of H2O2 up to two additional times after the initial pretreatment. These subsequent pretreatments were performed at 3-, 6-, or 12-h intervals after the initial pretreatment. Cells were harvested 24-h after initial treatment. Samples were then lysed and proteolytic capacity was measured through degradation of SucLLVY-AMC. Values are means 7 SE, where n ¼ 3. (B) Up to three additional treatments of H2O2 can be performed on cells with no reduction in the adaptive increase in proteolytic capacity, provided a 12-h recovery time is permitted between treatments. Cells were prepared and pretreated as for Fig. 1. Some cells were then pretreated with the same dose of H2O2 for up to three additional times after the initial pretreatment at 12-h intervals. Cells were harvested 48-h after initial treatment. Samples were then lysed and proteolytic capacity was measured through degradation of Suc-LLVY-AMC. Values are means 7 SE, where n ¼ 3. Activity is plotted as micromoles of AMC released per minute per gram of lysate.

third pretreatments at only 3-h intervals, the adaptation was clearly strongly limited (Fig. 3A). Having seen that a second pretreatment 12-h after the initial pretreatment was tolerated we wished to test if further 12-h interval pretreatments would also be tolerated, or if there would be a limit to the number of 12-h pretreatments that could be borne; in essence, therefore, we actually studied the effects of chronically repeated stress by exposing cells to an adaptive dose of H2O2 and then pretreating the cells a further one to three times at 12-h intervals. At 48-h after the first pretreatment all cells were harvested and proteolytic capacity was measured by degradation of Suc-LLVY-AMC. Our results show that even four pretreatments at the adaptive dose of 1 nmol of H2O2 per 105 cells were tolerated and produced similar and consistent increases in proteolytic capacity (Fig. 3B). It should be noted that the results of Fig. 3A were obtained in experiments performed 24-h after initial H2O2 pretreatment, whereas the results of Fig. 3B were obtained 48-h after initial pretreatment. As shown in Fig. 2A, the adaptive effects of a single pretreatment are lost within 48-h, thus the 12-h interval repeated pretreatments actually extended the adaptation. In contrast to the 12-h-interval results of Fig. 3B, the results of Fig. 3A clearly showed that H2O2 treatments at 3-h intervals had detrimental effects on adaptation, but the 6-h-interval data were somewhat equivocal. We decided to further study the effects of multiple 6-h-interval H2O2 treatments on the capacity of cells to degrade an actual protein (rather than the Suc-LLVY-AMC peptide model substrate) and for this we used both normal and oxidized hemoglobin, as previously described [3,4,18,40]. The capacity of cell extracts to selectively degrade Hbox rose after a single H2O2 pretreatment and was unaffected by a second pretreatment, but was reduced with a third successive H2O2 pretreatment (Fig. 4A).

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Fig. 4. Adaptive effects of H2O2 pretreatments at 6-h intervals. (A) The blunting of adaptive response with 6-h repeated H2O2 treatments appears to reduce not just proteolytic capacity, but also the adaptive increase in capacity to selectively degrade oxidized proteins. Cells were prepared and pretreated as for Fig. 1. Some cells were then pretreated with the same dose of H2O2 up to three additional times after the initial pretreatment, at 6-h intervals. Cells were harvested 24-h after initial treatment. Samples were then lysed and proteolytic capacity was measured through degradation of Hb–AMC or Hbox–AMC as substrate. Values are means 7 SE, where n ¼ 6. The inset shows the ratio of degradation of Hbox to degradation of Hb and reveals that the selective degradation of oxidized Hb actually declined significantly after both the second and the third H2O2 pretreatments. Activity is plotted as nanomoles of AMC released per minute per gram of lysate. (B) Repeated stress at 6-h intervals appears to blunt the adaptive increase in oxidative stress tolerance as well as blunting the increase in proteolytic capacity. Cells were prepared and treated as for (A) and then challenged with a toxic dose of H2O2, which was 500 mmol of H2O2 per 105 cells (1 mM H2O2 final concentration), 24-h after pretreatment. Cell counts were taken 24-h after H2O2 challenge. Values are plotted as a percentage of the cell counts of samples that were not exposed to the challenge and are shown as means 7 SE, where n ¼ 6. Values are plotted as % of unchallenged controls.

Interestingly, the capacity to degrade native Hb plateaued after two pretreatments, but did not decline even after the third successive treatment. Thus, the selective, preferential degradation of Hbox over that of native Hb actually declined significantly after both the second and the third H2O2 pretreatments (Fig. 4A, inset). We also tested tolerance to H2O2 stress, in the same cells, using our pretreatment/challenge model described for Figs. 1 and 2. We observed an increase in oxidative stress tolerance with a single pretreatment, which progressively declined with subsequent pretreatments (Fig. 4B). From this we can conclude that repeated H2O2 pretreatments within in a short time frame (6-h) not only reduced the adaptive increase in proteolytic capacity to degrade oxidized proteins, but also diminished the adaptive increase in oxidative stress tolerance. Having found that repeated H2O2 pretreatments at 3- or 6-h intervals were detrimental to the adaptive response (Figs. 3A, 4A, and 4B), but that pretreatments at 12-h intervals still allowed a full adaptive increase in proteolytic capacity, we wanted to determine if pretreatments at 12-h intervals would also allow a full adaptive increase in tolerance to severe H2O2 challenge. In addition we were also interested in whether the concentration of H2O2 used in the repeated stresses would affect the response; in particular, we wanted to know whether lowering the pretreatment concentration might mitigate against the negative effects of repeated treatments. We found that the original adaptive dose of 1.0 nmol H2O2 per 105 cells did not diminish the increase in tolerance to oxidative stress for the first two treatments and had only a weakly detrimental effect after the third treatment. When we used lower H2O2 pretreatments (e.g., 10–100 pmol of H2O2 per 105 cells) than the normally optimal adaptive dose (of 1 nmol of

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proteasome, as seen with mammalian cell cultures. This was determined using a line containing RNAi against the prosb2 20S proteasome subunit. This line was crossed to the Act-GS-255B strain [45] enabling the RNAi to be conditionally expressed in the presence of the drug RU486 in all the somatic tissues of the adult fly [46]. Additionally, the Act-GS-255B strain was crossed with w[1118] flies as controls for potential effects of RU486. The female progeny of these crosses were then cultured 7RU486 for 2 days. Flies were then pretreated 7100 mM H2O2 for 8-h and then returned to vials 7RU486 for a further 16-h. The flies were then challenged by incubation in vials containing Kim-Wipes soaked in 1.0 ml of 5% sucrose plus 4.4 M H2O2. We found that H2O2 pretreatment caused an increase in oxidative stress tolerance in control flies, which was lost with proteasome RNAi knockdown (Figs. 6B and 6C). The role of H2O2 and proteasome in oxidative stress adaption in flies is explored in more detail in a previously published work [39]. It should be noted that the experiments of Fig. 6 are confirmatory, not duplicative, of our previous recent results [39].

cells/ml)

Fig. 5. Adaptive effects of H2O2 pretreatment at 12-h intervals. The adaptive increase in oxidative stress tolerance is not affected by repeated H2O2 treatments at the adaptive dose if it is performed at 12-h intervals. If, however, subadaptive treatments are performed, they become more effectively adaptive with repeated treatment. If higher pretreatment doses are used, then repeated pretreatments have a detrimental effect on oxidative stress tolerance. Cells were grown to 10% confluence and then pretreated with various doses of H2O2 at 0-, 12-, 24-, and 36-h after initial treatment. Samples were then challenged with 5m mol of H2O2 per 105 cells (1 mM H2O2 final concentration) for 1-h; 24-h later cell counts were taken. Values are plotted as a percentage of the cell counts of samples that were not exposed to the challenge and are shown as means 7 SE, where n ¼ 4.

H2O2 per 105 cells), the repeated treatments actually exerted a progressively beneficial effect on oxidative stress tolerance. Conversely, H2O2 treatments higher (e.g., 10 nmol of H2O2 per 105 cells) than the normal adaptive dose had a strongly detrimental effect on oxidative stress tolerance (Fig. 5). From these results we conclude that repeated H2O2 pretreatments are permissive of oxidative stress adaptation as long as the interval of repeated treatment is 12-h or more, or if the level of H2O2 pretreatment is lowered. Role of the proteasome in oxidative stress adaptation We have previously shown in mammalian cells [3,4,33,41] that the adaptive response to a mild oxidant pretreatment is strongly dependent on Nrf2-induced increases in expression of the 20S proteasome and the proteasome regulators Pa28ab and Pa28g. There have been several reports that mild oxidant exposure of the model organism D. melanogaster (the common fruit fly) will also cause an adaptive increase in oxidative stress tolerance (Fig. 6C and [39,42–44]). It was, therefore, important to test whether, as in mammalian cells, a mild H2O2 pretreatment would lead to an increase in both proteolytic capacity and oxidative stress in fruit flies. Although we have recently investigated this question in both flies and worms (with positive results), the reviewers of the current paper (and the journal’s associate editor) specifically wanted to us to test again for a causal relationship between increased proteasome-dependent proteolytic capacity and increased oxidative stress resistance under the exact conditions of the current experiments. To test this we pretreated flies with various concentrations of H2O2 pretreatments (as done previously [39,43]) and then measured proteolytic capacity 18-h later. We observed that H2O2 pretreatment caused a two- to four-fold increase in proteolytic capacity (Fig. 6A). Next we wished to test if the H2O2-induced increase in proteolytic capacity was dependent upon increases in

Adaptive responses to multiple H2O2 pretreatments in D. melanogaster We next wanted to use the Drosophila model of oxidative stress adaptation to test whether repeated stress has effects at the organismal level similar to those at the cellular level. To test this we performed repeated stress adaptation by exposing flies to 8-h treatments of the adaptive dose of H2O2 zero, one, two, or three times. These treatments were performed at 1-, 3-, or 7-day intervals. Challenge was performed 16-h after the end of the final pretreatment. In all cases, flies exposed to a single pretreatment did notably better than control (non-pretreated) flies after the H2O2 challenge (Fig. 7A and B). With a second pretreatment just 1 day after the first treatment, however, all of the adaptive benefit was lost, and if a third treatment was given after just 1 more day the pretreated flies did much worse than controls. Even if 3-day intervals were allowed between pretreatments the flies exposed to a second or third pretreatment still had a severely blunted adaptive response. Finally, if a 7-day interval was allowed between pretreatments there was no notable difference between flies pretreated once, twice, or three times (Figs. 7A and 7B). From these results it is apparent that for whole flies, as for cultured mammalian cells, multiple pretreatments can be tolerated and still enable a full adaptive response, only with a sufficiently long recovery time between treatments. In Figs. 7A and 7B the assays in all cohorts were started 4–6 days after maturation. Because of the difference in pretreatment intervals, however, flies in the 1-day pretreatment interval cohort were challenged at 7–9 days after maturation, whereas flies in the 3-day pretreatment interval cohort were challenged at 13–15 days after maturation, and flies in the 7-day pretreatment interval cohort were challenged at 25–27 days after maturation. As has been shown previously [47] there is an age-dependent decline in oxidative stress tolerance over the time frame we studied. We also saw this age-associated decline in oxidative stress tolerance with 7- to 9-day-old flies surviving an average of 54-h after H2O2 challenge, whereas 25- to 27-day-old flies survived an average of only 38-h after H2O2 challenge: a 30% decline in baseline stress tolerance. The older flies were able to mount a significant adaptive response to H2O2 pretreatment but even the most adapted older flies still did not quite reach the median survival times exhibited by the control young flies (compare Fig. 7B, right, with the control values in Fig. 7B, left). This suggests that perhaps one of the factors contributing to age-associated deterioration might be a reduced ability to respond to changes in stresses, such as oxidative stress.

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Fig. 6. Adaptation of D. melanogaster to acute H2O2 pretreatment. (A) H2O2 treatment of D. melanogaster increases proteolytic capacity in females. Triplicate vials of w[1118] flies were pretreated by exposure to ½ a Kim-Wipe soaked in 1 ml of 5% sucrose plus the indicated concentrations of H2O2. Proteolytic capacity assays were performed on flies 24-h later as described under Materials and methods. Values are shown as nmol of AMC released/min/mg lysate. (B, C) H2O2 treatment of D. melanogaster increases oxidative stress tolerance, which is lost with knockdown of the proteasome subunit prosb2. (B) (#) w[1118]  (~) Act-GS-255B or (C) (#) Prosb2RNAi  (~) Act-GS-255B Drosophila. In all cases flies were cultured 7 RU486 for 2 days and then pretreated 7100 mM H2O2 (7 RU486) for 24-h. After pretreatment, the flies were returned to 7 RU486 vials for 16-h and then challenged by exposure to ½ a Kim-Wipe soaked in 1 ml of 5% sucrose plus 4.4 M H2O2. Values are plotted as means 7 SE, where n ¼ 3.

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Fig. 7. D. melanogaster adaptation to H2O2 challenge under repeated H2O2 pretreatment. (A) Like in cultured mammalian cells, H2O2 pretreatment increases Drosophila oxidative stress tolerance and this adaptation is blunted by repeated treatments, though the repeated treatments are tolerated if a sufficiently long recovery period (7 days) is permitted. Experiments were performed using vials of 20 4- to 6-day-old female w[1118] flies, which were cultured in vials containing normal food. Flies were incubated in vials containing ½ a Kim-Wipe soaked in 1 ml of 5% sucrose plus 100 mM H2O2 zero to three times for 6-h intervals. These pretreatments were preformed every day, every 3 days, or every 7 days. Then, 1 day after the final pretreatment, all flies were challenged by exposure to ½ a Kim-Wipe soaked in 1 ml of 5% sucrose plus a toxic dose of H2O2 (4.4 M). The flies were maintained in these vials and survival was scored every 8 h. Values are plotted as a percentage of population before challenge and are shown as means 7 SE, where n ¼ 3. (B) Values from (A) plotted as median survival time after initiation of H2O2 challenge, where n ¼ 60. (C) Repeated treatment, even when a long recovery time is permitted, appears to reduce fly life span. Flies were cultured in vials containing normal food. The flies were then transferred every day, every 3.5 days, or every 7 days to vials containing ½ a Kim-Wipe soaked in 1 ml of 5% sucrose plus 100 mM H2O2 for 8-h intervals. Fly survival was counted every 3.5 days and treatments were continued until all flies were dead. (D) Values from (C) plotted as median survival time after initiation of H2O2 challenge, where n ¼ 60.

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Fig. 8. Adaptation to chronic oxidative stress. (A) Chronic oxidative stress exposure of mammalian cell culture produces a stronger adaptive response than acute exposure with weaker oxidant concentrations, but produces a blunted response compared to acute exposures with higher oxidant concentrations. MEF cells were grown to 20% confluence and then treated with 10–1000 fg/ml glucose oxidase (GO) for either 1- or 24-h; the cells were then washed and permitted to adapt and recover for 24-h. Rates of hydrogen peroxide production were as follows: 1 fmol H2O2 per minute for 10 fg GO, 10 fmol H2O2 per minute for 100 fg GO, or 100 fmol H2O2 per minute for 1000 fg GO. After recovery, the cells were challenged with 500 mmol of H2O2 per 105 cells for 1-h, and cell counts were taken 24-h later. Values are plotted as a percentage of the cell counts of samples that were not exposed to the challenge and are shown as means 7 SE, where n ¼ 3. (B) Chronic oxidative stress exposure in Drosophila is beneficial up to a point, after which the adaptive response is blunted. The lower the dose of oxidant exposure, the longer the time for which chronic exposure is beneficial. Flies were cultured as for Fig. 5, with 4- to 6-day-old female w[1118] flies being pretreated with 0, 10, 100, or 1000 mM H2O2 for 8-, 24-, or 48-h. Flies were then challenged with a toxic dose of H2O2 (4.4 M) at 24-h after the end of the pretreatment. Flies were maintained in these vials and survival was scored every 8-h. Median survival time was determined, with n ¼ 60.

In addition to acute oxidative stress tolerance, we were also interested in whether repeated oxidative stress adaptation had any effects on the eventual life-span of flies. We considered this to be something of a measure of the long-term repercussions of oxidative stress adaptation. To explore this question we performed life-span assays on flies that were repeatedly exposed to oxidative stress. In these life-span assays, flies were either maintained as naı¨ve (control) flies or exposed to 8-h treatments of the same dose of H2O2 used for oxidative stress adaptation experiments above, either weekly, twice weekly, or daily (Figs. 7C and 7D). In these experiments we found that flies pretreated weekly, twice weekly, or daily all had a shorter life span than naı¨ve control flies, although the degree of reduction in life-span was roughly proportional to the frequency of exposure, with flies treated daily living the shortest time. Chronic H2O2 adaptation in MEF cells In addition to studying repeated oxidative stress adaptation, a limited set of experiments was performed under chronic oxidative stress conditions. Models of chronic H2O2 stress are particularly difficult because H2O2 and many other oxidants are rapidly degraded by cells. Thus, it is not possible to use a single H2O2 dose (without removal) to study chronic exposure in cell culture. One solution is the use of the enzyme glucose oxidase, which catalyzes the direct two-electron reduction of oxygen to H2O2, using reducing equivalents from the oxidation of glucose. We found that if a sufficiently low dose (10–1000 fg/ml) of glucose oxidase is used (in the presence of nonlimiting glucose), then glucose oxidase treatment can be maintained for 24-h with no significant effects on cell division rates for MEF cells. The rate of H2O2 generation by 10 fg/ml glucose oxidase was 1 fmol of H2O2 per minute. To compare the effects of acute vs chronic oxidant exposure on oxidative stress adaptation, we treated cells with glucose oxidase for either 1 (an acute stress adaptation) or for

24-h (our chronic stress adaptation). Cells were challenged 24-h after this treatment with a toxic dose of H2O2 and cell counts were taken another 24-h later. We found that an acute treatment with glucose oxidase had a progressively stronger adaptive effect on tolerance to oxidative stress in the range from 10 to 1000 fg/ml (Fig. 8A). In comparison, in the chronic cell treatment regime, 10 and 100 fg/ml treatment had stronger beneficial effects than the same dose used as an acute exposure. Chronic 24-h exposure to 1000 fg glucose oxidase, however, was clearly toxic for the MEF cells, which had a much weaker adaptive response than when the same glucose oxidase concentration was used for only a 1-h exposure (Fig. 8A). Chronic H2O2 adaptation in flies Similar results are seen using a fly model of chronic H2O2 adaptation (Fig. 8B). Flies were placed in vials containing KimWipes soaked in 5% sucrose 7 three potentially adaptive doses of H2O2 for 0-, 6-, 24-, or 48-h. The flies were then permitted to recover on normal food for 24 h and then transferred to challenge vials, which contained Kim-Wipes soaked in 5% sucrose and a toxic concentration of 4.4 M H2O2. Fly survival was then scored over the following 80-h. We found that when 100 mM H2O2 was used as an adaptive treatment, flies treated for 8-h were considerably more resilient to H2O2 challenge, whereas flies treated for 24-h were weakly more resilient and flies treated for 48-h had lost the adaptive response and were as sensitive as untreated flies. In comparison when a stronger concentration of 1 mM H2O2 was used as the adaptive treatment, then flies treated for 8-h were weakly more resistant to oxidative stress than control flies, whereas flies treated for 24- or 48-h were no more resilient than control flies. Finally, when a weaker treatment of 10 mM H2O2 was used for the adaptive treatment, then 6- and 24-h exposures had no beneficial effect on oxidative stress tolerance, but 48-h exposure had a weakly beneficial effect (Fig. 8B).

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Discussion In previous reports we have shown that a mild exposure of cells to H2O2 or other oxidants produces a transient increase in tolerance to oxidative stress. We have also shown that this transient adaptation is partly a product of enhanced capacity to remove oxidized proteins, which occurs through changes in 20S proteasome levels and activity [2–4,33]. These previous studies were, however, limited to acute oxidative stress adaptation rather than repeated or chronic stress, which might be more representative of ‘‘real-world’’ oxidative stress exposures. In the present investigation, we set out to study the ability of cells and flies to adapt to such repeated or chronic oxidative stress exposures. A single H2O2 pretreatment increased the tolerance of MEF cells to a subsequent H2O2 challenge (as measured though cell counts, BrdU incorporation, proteolytic capacity, and protein carbonyl assays) and this was blocked in cells also treated with an siRNA against the proteasome b1 subunit, to prevent increases in proteasome levels. In contrast with these results for an acute H2O2 exposure, repeated H2O2 pretreatments with short time intervals (3–6 h) generally blunted the adaptive response. Two modifications to the MEF cell repeated H2O2 pretreatment protocol were effective in permitting an improved adaptive response: increasing the intervals between H2O2 treatments to 12-h or decreasing the H2O2 concentration. When 12-h intervals between successive H2O2 treatments were used, at the same H2O2 concentration used for acute adaptation, the MEF cell adaptive response was actually prolonged to at least 48-h. Similarly, a concentration of H2O2 (5 mM) only 10% of that normally used became a highly effective adaptive treatment, with effects lasting at least 48-h, when used for multiple treatments at 12-h intervals. Proteolytic capacity and tolerance to oxidative stress tracked very well with one another in these MEF cell experiments, again emphasizing the close relationship between capacity to remove oxidatively damaged proteins and capacity to survive an oxidative stress [3,33]. Using an H2O2 pretreatment/challenge model we found that Drosophila are capable of a strong H2O2-induced transient adaptive increase in oxidative stress tolerance. Proteolytic capacity and tolerance to oxidative stress again tracked very well with one another in these Drosophila experiments, with RNAi experiments showing that blocking adaptive increases in proteasome levels also blocked adaptive increases in survival, once more emphasizing the close relationship between capacity to remove oxidatively damaged proteins and capacity to survive an oxidative stress. Also, as in MEF cells, the increased resistance of flies to oxidative stress challenge was blunted by repeated H2O2 exposures at short intervals (1–3 days), although if a sufficiently long interval was permitted between treatments (7 days), then the repeated treatments appeared to be tolerated and generated a significant adaptive increase in oxidative stress tolerance. Conversely, however, all repeated treatments appeared to have a negative long-term effect, because flies that were repeatedly exposed to H2O2 had a shorter life spans than untreated flies. We also observed a progressive decline in Drosophila life span with frequency of H2O2 treatment. When MEF cells were chronically exposed to continuous H2O2 production, generated by the enzyme glucose oxidase, we actually observed an enhanced adaptive response (H2O2 resistance) at low rates of H2O2 generation but an almost complete loss of stress adaptation at higher rates of H2O2 production. In Drosophila, chronic H2O2 exposure had almost universally negative effects, with flies exhibiting little to no adaptive response after 24- or 48-h. In conclusion for MEF cells—repeated MEF cell oxidant exposures seem to prevent adaptive stress responses if the interval between exposures is too short and/or if each exposure is at too high an oxidant level. In contrast, repeated cell exposures at 12-h

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intervals, especially at lower oxidant concentrations, seem to potentiate positive stress adaptations and extend the period of protection. Chronic oxidant exposure at low oxidant levels can actually potentiate and extend adaptive responses, but chronic exposure to higher oxidant levels prevents adaptation. In conclusion for D. melanogaster—repeated oxidative stress adaptation at intervals of 1 or 3 days is toxic but intervals of 7 days are tolerated in terms of short-term survival. Life span, however, seems to be negatively influenced by all repeated stress adaptation regimens. Short-term survival is also negatively influenced by chronic oxidative stress adaptation at all levels.

Acknowledgments This research was supported by Grant ES03598 and by ARRA Supplement 3RO1-ES 003598-22S2, both from the NIH/NIEHS to K.J.A.D., and a grant from the Department of Health and Human Services (NIH/NIA) to J.T. (AG011833). References [1] Burdon, R. H Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic. Biol. Med 18:775–794; 1995. [2] Wiese, A. G.; Pacifici, R. E.; Davies, K. J. Transient adaptation of oxidative stress in mammalian cells. Arch. Biochem. Biophys. 318:231–240; 1995. [3] Pickering, A. M.; Koop, A. L.; Teoh, C. Y.; Ermak, G.; Grune, T.; Davies, K. J. The immunoproteasome, the 20S proteasome and the PA28alphabeta proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem. J. 432:585–594; 2010. [4] Pickering, A. M.; Linder, R. A.; Zhang, H.; Forman, H. J.; Davies, K. J. Nrf2dependent induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress. J. Biol. Chem. 287:10021–10031; 2012. [5] Davies, K. J. Protein damage and degradation by oxygen radicals. I. General aspects. J. Biol. Chem. 262:9895–9901; 1987. [6] Davies, K. J. Protein modification by oxidants and the role of proteolytic enzymes. Biochem. Soc. Trans 21:346–353; 1993. [7] Davies, K. J. Oxidative stress: the paradox of aerobic life. Biochem. Soc. Symp. 61:1–31; 1995. [8] Davies, K. J. An overview of oxidative stress. IUBMB Life 50:241–244; 2000. [9] Davies, K. J.; Delsignore, M. E. Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure. J. Biol. Chem. 262:9908–9913; 1987. [10] Davies, K. J.; Delsignore, M. E.; Lin, S. W. Protein damage and degradation by oxygen radicals. II. Modification of amino acids. J. Biol. Chem. 262:9902–9907; 1987. [11] Salo, D. C.; Pacifici, R. E.; Lin, S. W.; Giulivi, C.; Davies, K. J. Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. J. Biol. Chem. 265:11919–11927; 1990. [12] Giulivi, C.; Davies, K. J. Dityrosine and tyrosine oxidation products are endogenous markers for the selective proteolysis of oxidatively modified red blood cell hemoglobin by (the 19 S) proteasome. J. Biol. Chem 268:8752–8759; 1993. [13] Pacifici, R. E.; Kono, Y.; Davies, K. J. Hydrophobicity as the signal for selective degradation of hydroxyl radical-modified hemoglobin by the multicatalytic proteinase complex, proteasome. J. Biol. Chem 268:15405–15411; 1993. [14] Habig, W. H.; Pabst, M. J.; Jakoby, W. B. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249:7130–7139; 1974. [15] Meister, A. Glutathione–ascorbic acid antioxidant system in animals. J. Biol. Chem. 269:9397–9400; 1994. [16] McCord, J. M.; Fridovich, I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6049–6055; 1969. [17] Wood, Z. A.; Schroder, E.; Robin Harris, J.; Poole, L. B. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28:32–40; 2003. [18] Davies, K. J. Degradation of oxidized proteins by the 20S proteasome. Biochimie 83:301–310; 2001. [19] Ding, Q.; Dimayuga, E.; Keller, J. N. Proteasome regulation of oxidative stress in aging and age-related diseases of the CNS. Antioxid. Redox Signaling 8:163–172; 2006. [20] Grune, T.; Reinheckel, T.; Joshi, M.; Davies, K. J. Proteolysis in cultured liver epithelial cells during oxidative stress: role of the multicatalytic proteinase complex, proteasome. J. Biol. Chem. 270:2344–2351; 1995. [21] Chondrogianni, N.; Stratford, F. L.; Trougakos, I. P.; Friguet, B.; Rivett, A. J.; Gonos, E. S. Central role of the proteasome in senescence and survival of human fibroblasts: induction of a senescence-like phenotype upon its inhibition and resistance to stress upon its activation. J. Biol. Chem 278:28026–28037; 2003.

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